Mechanistic basis for co-operativity in kinesin-1 / cargo recognition

Cells possess many specialised components that must be in the right place at the right time to fulfil their function. After their use, these components must be transported away for recycling or degradation. Mis-regulation or disruption of these transport processes can contribute to many human diseases ranging from neurodegenerative conditions such as Alzheimer's disease to cancer and even contribute to viral infections by HIV-1 or bacterial infections such as Salmonella. To move components around, cells use a transport system composed of a network of cables known as the microtubule network. Much like a railway network, these cables link together regions of the cell. Cells possess vehicles that travel along this network known as molecular motors, of which our proposed motor of study, kinesin-1, is one of the most important. These motors can selectively attach to cellular components and move them on the microtubule network. They can also control the organisation of the network itself by sliding against one another. Despite their importance across so many areas of cell biology, we lack a proper understanding of how these motors recognise the cargo that they carry and how this in turn controls the behaviour of the motor.

This joint proposal stems from a sustained successful partnership between the Dodding group, now at the School of Biochemistry of the University of Bristol and the Steiner group of the Randall Centre of Cell and Molecular Biophysics, King's College London. To date, their fruitful collaborative work has highlighted important aspects of how kinesin-1 attaches to the cellular components it carries (using protein-peptide interactions) and how these connections in turn control how kinesin-1 moves. Importantly, they have used this knowledge to identify small molecules (drug-like chemicals) that have allowed us to directly manipulate this system in cells for the first time.

These exciting findings have now promoted a series of new questions that will be addressed here. Until now, our studies have focused on defining quite simple and straightforward connections between kinesin-1 and its cargoes. However, it is becoming clear that this is only part of the picture and that this process requires multiple connections between kinesin-1 and its cargoes that work together. We propose that the precise nature of these 'co-operative' connections determines how transport works. Here we will explore how kinesin-1 can interact directly with the surface of membrane bound organelles (by attaching to membrane lipids) and how these connections work together with those we have already defined. Moreover, collections of proteins can interact with each other and kinesin-1 - we will seek to define those connections on a molecular level. We will continue to use the knowledge we acquire to further chemically manipulate this cargo attachment system to see if we can target specific aspects of kinesin-1 function. It is possible that in the long term, this may show how we can develop drugs to target kinesin-1 in human disease.

The microtubule motor kinesin-1 plays a central role in cell biology and pathology by virtue of its capacity to interact with many cellular components and transport them on the microtubule network. An understanding of how kinesin-1 interacts with its cargoes is crucial to understanding how cells are organised an space and time. Our recent work has defined key pathways that mediate cargo recognition and kinesin-1 activation and showing for the first time how we can chemically manipulate the activity of this enzyme in cells. Here, we will test the hypothesis that there are multiple pathways to recognition and activation dependent upon site(s) of cargo binding that rely on co-operative networks of motor-cargo interactions. These networks tailors kinesin-1 activity for specific transport functions. In this proposal, we will define and dissect those pathways and further seek to chemically manipulate them to test the mechanistic models that emerge. We will determine how the variable carboxy terminal domains of the kinesin-1 light chains (KLC) interact with cargo and how these interactions work together this those we have recently defined (Objective 1). We will establish the structural basis for the formation of JIP1/JIP3/KLC ternary complexes with the kinesin-1 light chains using X-ray crystallography and cryo-electron microscopy techniques and determine how these interactions control kinesin-1 activity (Objective 2). We will establish the structural basis for interaction of KLC with a novel small molecule activator of kinesin-1 and seek identify novel molecules that can chemically separate distinct kinesin-1 cargo recognition/activation pathways (Objective 3). Collectively, these goals will develop a more complete mechanistic understanding of fundamental principles underlying kinesin-1 cargo recognition, provide a functional basis for the isotype and isoform diversity within the family, and show how we might consider manipulating these mechanisms for therapeutic purposes

Due to the central importance of microtubule motor proteins for all forms of eukaryotic life, a core understanding of their basic function will impacts across a wide breadth of biomedical science.

i. Pharmaceutical and biotechnology industries
Work described in the proposal examines the molecular interface between kinesin-1 and cellular, viral and bacterial proteins associated with human diseases. These include Salmonella and poxvirus infections as well as neurological conditions such as Alzheirmer's Disease and Hereditary Spastic Paraplegia. As well as providing basic knowledge that is essential for an understanding of these diseases, an important aim of our research in the long term and for which this proposal will lay an important foundation is to determine whether manipulation of this interface could selectively control transport of cellular cargoes associated with human diseases. Our recent work suggests that chemical manipulation of these interfaces is indeed possible. This may lead to the commericalisation of the scientific knowledge obtained from this proposal and/or the formation of spin out companies, thereby contributing toward wealth creation and economic prosperity of the nation. This would also serve to attract R&D investment from global business.

ii. Patients who suffer from diseases where microtubule transport is impaired or usurped and clinicians who treat those diseases
As indicated above, we our work address a crucial questions that lies at the heart of cell biology and disease. Whether this involves the hijacking of the transport system in the case of pathogen infection or whether dysregulation of transport is an important consequence of disease as in the case of Alzheimer's, our research of these molecular interfaces and the chemical manipulation of them will add to the knowledge of understanding of these diseases and offers the long term hope of targeting the motor/cargo interface which could benefit many.

iii. The wider public
The wider public will gain from an increased understanding of how the human body works on a molecular level. The engagement of the public with academic science has become a priority and we will take steps as outlined in pathways to impact to ensure that our work is communicated to the widest non-academic audience possible.

iv Staff funded by this project
Staff working on this project will to receive a through training in molecular, cellular, and structural biology in one of the counties leading scientific institutions. It is highly likely that they will use this knowledge to make further contributions either in academic or industry, which will in turn benefit the UK economy. Moreover, this project will support the continued establishment of the Dodding Lab at Bristol.

Details of how we propose to maximize these impacts for these beneficiaries are described in our 'pathways to impact statement'.